Mammalian target of rapamycin activity is required for expansion of CD34+ hematopoietic progenitor cells

Background

The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic syndromes, suggesting that correct regulation of mammalian target of rapamycin is critical for normal hematopoiesis.

Design and Methods

An ex vivo granulocyte differentiation system was utilized to investigate the role of mammalian target of rapamycin in the regulation of myelopoiesis.

Results

Inhibition of mammalian target of rapamycin activity, with the pharmacological inhibitor rapamycin, dramatically reduced hematopoietic progenitor expansion, without altering levels of apoptosis or maturation. Moreover, analysis of distinct hematopoietic progenitor populations revealed that rapamycin treatment inhibited the expansion potential of committed CD34⁺ lineage-positive progenitors, but did not affect early hematopoietic progenitors. Further examinations showed that these effects of rapamycin on progenitor expansion might involve differential regulation of protein kinase B and mammalian target of rapamycin signaling.

Conclusions

Together, these results indicate that mammalian target of rapamycin activity is essential for expansion of CD34⁺ hematopoietic progenitor cells during myelopoiesis. Modulation of the mammalian target of rapamycin pathway may be of benefit in the design of new therapies to control hematologic malignancies.     

Varicella-zoster virus glycoproteins B and E are major targets of CD4+ and CD8+ T cells reconstituting during zoster after allogeneic transplantation

Background

After allogeneic hematopoietic stem-cell transplantation patients are at increased risk for herpes zoster as long as varicella-zoster virus specific T-cell reconstitution is impaired. This study aimed to identify immunodominant varicella-zoster virus antigens that drive recovery of virus-specific T cells after transplantation.

Design and Methods

Antigens were purified from a varicella-zoster virus infected cell lysate by high-performance liquid chromatography and were identified by quantitative mass spectrometric analysis. To approximate in vivo immunogenicity for memory T cells, antigen preparations were consistently screened with ex vivo PBMC of varicella-zoster virus immune healthy individuals in sensitive interferon-γ ELISpot assays. Candidate virus antigens identified by the approach were genetically expressed in PBMC using electroporation of in vitro transcribed RNA encoding full-length proteins and were then analyzed for recognition by CD4⁺ and CD8⁺ memory T cells.

Results

Varicella-zoster virus encoded glycoproteins B and E, and immediate early protein 62 were identified in immunoreactive lysate material. Predominant CD4⁺ T-cell reactivity to these proteins was observed in healthy virus carriers. Furthermore, longitudinal screening in allogeneic stem-cell transplantation patients showed strong expansions of memory T cells recognizing glycoproteins B and E after onset of herpes zoster, while immediate early protein 62 reactivity remained moderate. Reactivity to viral glycoproteins boosted by acute zoster was mediated by both CD4⁺ and CD8⁺ T cells.

Conclusions

Our data demonstrate that glycoproteins B and E are major targets of varicella-zoster virus specific CD4⁺ and CD8⁺ T-cell reconstitution occurring during herpes zoster after allogeneic stem-cell transplantation. Varicella-zoster virus glycoproteins B and E might form the basis for novel non-hazardous zoster subunit vaccines suitable for immunocompromised transplant patients.     

Cytokine-induced killer cells for cell therapy of acute myeloid leukemia: improvement of their immune activity by expression of CD33-specific chimeric receptors

Background

Cytokine-induced killer cells are ex vivo-expanded cells with potent antitumor activity. The infusion of cytokine-induced killer cells in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplant is well tolerated, but limited clinical responses have been observed. To improve their effector functions against acute myeloid leukemia, we genetically modified cytokine-induced killer cells with chimeric receptors specific for the CD33 myeloid antigen.

Design and Methods

SFG-retroviral vectors coding for anti-CD33-ζ and anti-CD33-CD28-OX40-ζ chimeric receptors were used to transduce cytokine-induced killer cells. Transduced cells were characterized in vitro for their ability to lyse leukemic targets (4-hour ⁵¹chromium-release and 6-day co-cultures assays on human stromal mesenchymal cells), to proliferate (³H-thymidine-incorporation assay) and to secrete cytokines (flow cytomix assay) after contact with acute myeloid leukemia cells. Their activity against normal CD34⁺ hematopoietic progenitor cells was evaluated by analyzing the colony-forming unit capacity after co-incubation.

Results

Cytokine-induced killer cells were efficiently transduced with the anti-CD33 chimeric receptors, maintaining their native phenotype and functions and acquiring potent cytotoxicity (up to 80% lysis after 4-hour incubation) against different acute myeloid leukemia targets, as also confirmed in long-term killing experiments. Moreover, introduction of the anti-CD33 chimeric receptors was accompanied by prominent CD33-specific proliferative activity, with the release of high levels of immunostimulatory cytokines. The presence of CD28-OX40 in chimeric receptor endodomain was associated with a significant amelioration of the anti-leukemic activity of cytokine-induced killer cells. Importantly, even though the cytokine-induced killer cells transduced with anti-CD33 chimeric receptors showed toxicity against normal hematopoietic CD34⁺ progenitor cells, residual clonogenic activity was preserved.

Conclusions

Our results indicate that anti-CD33 chimeric receptors strongly enhance anti-leukemic cytokine-induced killer cell functions, suggesting that cytokine-induced killer cells transduced with these molecules might represent a promising optimized tool for acute myeloid leukemia immunotherapy.     

Recipient lymphocyte infusion in MHC-matched bone marrow chimeras induces a limited lymphohematopoietic host-versus-graft reactivity but a significant antileukemic effect mediated by CD8+ T cells and natural killer cells

Background

Challenge of MHC-mismatched murine bone marrow chimeras with recipient-type lymphocytes (recipient lymphocyte infusion) produces antileukemic responses in association with rejection of donor chimerism. In contrast, MHC-matched chimeras resist eradication of donor chimerism by recipient lymphocyte infusion. Here, we investigated lymphohematopoietic host-versus-graft reactivity and antileukemic responses in the MHC-matched setting, which is reminiscent of the majority of clinical transplants.

Design and Methods

We challenged C3H→AKR radiation chimeras with AKR-type splenocytes (i.e. recipient lymphocyte infusion) and BW5147.3 leukemia cells. We studied the kinetics of chimerism using flowcytometry and the mechanisms involved in antileukemic effects using in vivo antibody-mediated depletion of CD8⁺ T and NK cells, and intracellular cytokine staining.

Results

Whereas control chimeras showed progressive evolution towards high-level donor T-cell chimerism, recipient lymphocyte infusion chimeras showed a limited reduction of donor chimerism with delayed onset and long-term preservation of lower-level mixed chimerism. Recipient lymphocyte infusion chimeras nevertheless showed a significant survival benefit after leukemia challenge. In vivo antibody-mediated depletion experiments showed that both CD8⁺ T cells and NK cells contribute to the antileukemic effect. Consistent with a role for NK cells, the proportion of IFN-γ producing NK cells in recipient lymphocyte infusion chimeras was significantly higher than in control chimeras.

Conclusions

In the MHC-matched setting, recipient lymphocyte infusion elicits lymphohematopoietic host-versus-graft reactivity that is limited but sufficient to provide an antileukemic effect, and this is dependent on CD8⁺ T cells and NK cells. The data indicate that NK cells are activated as a bystander phenomenon during lymphohematopoietic T-cell alloreactivity and thus support a novel type of NK involvement in anti-tumor responses after post-transplant adoptive cell therapy.     

Ex vivo expansion of hematopoietic progenitor cells is associated with downregulation of α4 integrin- and CXCR4-mediated engraftment in NOD/SCID β2-microglobulin-null mice

Background

Several studies indicate that ex vivo cytokine-supported expansion induces defective hematopoietic stem cell engraftment. We investigated the role of α4 integrin, α5 integrin and CXCR4 in engraftment of unmanipulated and cytokine-treated human cord blood CD34⁺ cells.

Design and Methods

Uncultured or expanded CD34⁺ cells were infused in NOD/SCID-β₂microglobulin-null mice. The function of α4, and α5 integrins and CXCR4 was assessed by incubating cells with specific neutralizing antibodies, prior to transplant. The activation state of α4 integrin was further tested by adhesion and migration assays.

Results

Neutralization of either α4 integrin or CXCR4 abolished engraftment of uncultured CD34⁺ cells at 6 week spost-transplant, while α5 integrin neutralization had no significant effect. However, after short-term ex vivo culture, blocking α4 integrin or CXCR4 did not affect repopulating activity whereas neutralization of α5 integrin inhibited engraftment. Using soluble vascular cell adhesion molecule-1 binding assays, we observed that α4 integrin affinity in fresh CD34⁺ cells was low and susceptible to stimulation while in cultured CD34⁺ cells, it was high and insensitive to further activation. In addition, stromal cell-derived factor-1 stimulated migration across vascular cell adhesion molecule-1 in fresh CD34⁺ cells but not in cultured CD34⁺ cells.

Conclusions

Our data show that ex vivo culture of hematopoietic progenitor cells is associated with downregulation of both α4 integrin- and CXCR4-mediated engraftment. Further investigations suggest that this is caused by supraphysiological increase of α4 integrin affinity, which impairs directional migration across vascular cell adhesion molecule-1 in response to stromal cell-derived factor-1. Such changes may underlie the engraftment defect of cytokine-stimulated CD34⁺ cells.     

Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms

Background

CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (α-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy.

Design and Methods

We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and α-GalCer in the treatment of mice engrafted with CD1d⁺ lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice.

Results

The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence α-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and α-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d⁺ masses. In addition, CD1d-restricted T-cell treatment plus α-GalCer eradicated small C1R-CD1d⁺ nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules.

Conclusions

Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and α-GalCer may represent a new immunotherapeutic tool for treatment of CD1d⁺ hematologic malignancies.     

The Fusion Protein of CTP-HBcAg18-27-Tapasin Mediates the Apoptosis of CD8+T Cells and CD8+ T Cell Response in HLA-A2 Transgenic Mice

Background:

HBV-specific cytotoxic T lymphocyte (CTL) activity is believed to play a critical role in controlling HBV infection. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway manipulates cell fate decisions in many different cell types by regulating the activity of downstream effectors. We have previously testified that the fusion protein of CTP-HBcAg18-27--Tapasin could enter the cytoplasm of dendritic cells and efficiently induce robust specific CTL response in vitro.

Objectives:

In the present study, we evaluated specific CTL response and the level of apoptosis of CD8+ T cells induced by CTP-HBcAg18-27-Tapasin in HLA-A2 transgenic mice (H-2Kb). Meanwhile, we preliminary investigated PI3K, phosphorylation level of Akt, and mammalian target of rapamycin (mTOR) as positive regulator of the magnitude and effector function of the hepatitis B virus-specific cytotoxic T lymphocytes in HLA-A2 transgenic mice.

Materials and Methods:

HLA-A2 transgenic mice were immunized by intramuscular injection in the hind legs three times at one-week intervals with PBS, CTP-HBcAg18-27-Tapasin (50 μg), CTP-HBcAg18-27 (50 μg), HBcAg18-27-Tapasin (50 μg), and HBcAg18-27 (50 μg). One week after the last immunization, mice were sacrificed and splenocytes were harvested in strile condition. The specific CTL response was analyzed by flow cytometry and enzyme linked immunosorbent assay (ELISA); the expression of (PI3K)/Akt signaling was detected by RT-PCR and western blot.

Results:

The results showed that CTP-HBcAg18-27-Tapasin significantly increased the percentages of IFN-γ⁺ CD8α⁺ T cells, the numbers of these polyfunctional triple-cytokine-producing (IFN-γ, TNF-α, and IL-2) CD8⁺T cells, the secretion of cytokine IFN-γ, IL-2, and TNF-α, while in comparison to control group, it significantly decreased the percentage of apoptotic CD8⁺ T cells in HLA-A2 transgenic mice. Moreover, the expression of PI3K, P-Akt, and P-mTOR was significantly upregulated in CTP-HBcAg18-27-Tapasin group compared with control groups.

Conclusions:

In conclusion, CTP-HBcAg18-27-Tapasin could reduce apoptosis of CD8⁺ T cells, increase the percentages of IFN-γ⁺ CD8α⁺ T cells, and elicit cell-mediated immunity in HLA-A2 transgenic mice; these processes were associated with activation of the PI3K/Akt signaling pathway.     

Prolonged ex vivo culture of human bone marrow mesenchymal stem cells influences their supportive activity toward NOD/SCID-repopulating cells and committed progenitor cells of B lymphoid and myeloid lineages

Background

Bone marrow mesenchymal stem cells support proliferation and differentiation of hematopoietic progenitor cells in vitro. Since these cells constitute a rare subset of bone marrow cells, mesenchymal stem cell preparations for clinical purposes require a preparative step of ex vivo multiplication. The aim of our study was to analyze the influence of culture duration on mesenchymal stem cell supportive activity.

Design and Methods

Mesenchymal stem cells were expanded for up to ten passages. These cells and CD34+ cells were seeded in cytokine-free co-cultures after which the phenotype, clonogenic capacity and in vivo repopulating activity of harvested hematopoietic cells were assessed.

Results

Early passage mesenchymal stem cells supported hematopoietic progenitor cell expansion and differentiation toward both B lymphoid and myeloid lineages. Late passage mesenchymal stem cells did not support hematopoietic progenitor cell and myeloid cell outgrowth but maintained B-cell supportive ability. In vitro maintenance of NOD/SCID mouse repopulating cells cultured for 1 week in contact with mesenchymal stem cells was effective until the fourth passage of the mesenchymal cells and declined thereafter. The levels of engraftment of CD34⁺ cells in NOD/SCID mice was higher when these cells were co-injected with early passage mesenchymal stem cells; however mesenchymal cells expanded beyond nine passages were ineffective in promoting CD34⁺ cell engraftment. Non-contact cultures indicated that mesenchymal stem cell supportive activity involved diffusible factors. Among these, interleukins 6 and 8 contributed to the supportive activity of early passage mesenchymal stem cells but not to those of late passage cells. The phenotype, as well as fat, bone and cartilage differentiation capacity, of mesenchymal stem cells did not change during their culture.

Conclusions

Extended culture of mesenchymal stem cells alters the ability of these cells to support hematopoietic progenitor cells without causing concomitant changes in their phenotype or differentiation capacity.     

The Expression of Programmed Death-1 in Circulating CD4+ and CD8+ T Cells during Hepatitis B Virus Infection Progression and Its Correlation with Clinical Baseline Characteristics

Background/Aims

Programmed death-1 (PD-1) expression was investigated in CD4⁺ and CD8⁺ T cells from hepatitis B virus (HBV)-infected patients at the chronic hepatitis B (CHB) infection, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) stages.

Methods

PD-1 expression in circulating CD4⁺ and CD8⁺ T cells was detected by flow cytometry. The correlations between PD-1 expression and HBV viral load, alanine aminotransaminase (ALT) levels and aspartate aminotransferase (AST) levels were analyzed using GraphPad Prism 5.0.

Results

PD-1 expression in CD4⁺ and CD8⁺ T cells was significantly increased in both the CHB group and advanced-stage group (LC plus HCC). In the CHB group, PD-1 expression in both CD4⁺ and CD8⁺ T cells was positively correlated with the HBV viral load, ALT, and AST levels. However, in the LC plus HCC group, significant correlations between PD-1 expression and the clinical parameters were nearly absent.

Conclusions

PD-1 expression in peripheral CD4⁺ and CD8⁺ T cells is dynamic, changes with HBV infection progression, and is related to HBV viral load and liver function, especially in CHB. PD-1 expression could be utilized as a potential clinical indicator to determine the extent of virus replication and liver injury.     

Depletion of T regulatory cells through selection of CD127-positive cells results in a population enriched in memory T cells: implications for anti-tumor cell therapy

Background

Donor lymphocyte infusions can induce remissions in patients with relapse after allogeneic hematopoietic stem cell transplantation. Nevertheless, some grafted patients never display any signs of alloreactivity, either following allogeneic hematopoietic stem cell transplantation or after donor lymphocyte infusions. Consequently, they do not develop graft-versus-host disease and frequently do not respond to donor lymphocyte infusions. In a recently published clinical trial, we observed that elimination of CD4⁺CD25⁺Foxp3⁺ natural regulatory T cells from the donor lymphocyte product could improve alloreactivity and the associated anti-tumor effect in a small proportion of patients with relapsed hematologic malignancies. Here, we aimed to improve the effect of donor lymphocyte infusion by modifying the procedure for depletion of T regulatory cells.

Design and Methods

We directly compared depletion of regulatory T cells from human peripheral blood mononuclear cells achieved by selection of CD127-positive cells or by selection of CD25-negative cells. We tested the manipulated products (i) in vitro in mixed lymphocyte reactions and against pathogen-derived recall antigens and (ii) in vivo in experimental graft-versus-host disease.

Results

In vitro, we found that depletion of regulatory T cells through CD127 positive selection improved both alloreactive and pathogen-specific immune responses. In vivo, we observed accelerated donor T-cell division and enhanced graft-versus-host disease due to efficient regulatory T-cell depletion accompanied by enrichment in memory T cells.

Conclusions

Our results show that the strategy of CD127 positive selection is an efficient way of eliminating regulatory T cells from donor lymphocyte infusions and improves alloreactivity. This supports the investigation of CD127 positive selection in place of elimination of CD25-positive cells for clinical applications.Key words: CD127 positive selection, Treg, alloreactivity, donor lymphocyte infusion     

Repeated PR1 and WT1 peptide vaccination in Montanide-adjuvant fails to induce sustained high-avidity, epitope-specific CD8+ T cells in myeloid malignancies

Background

We previously showed that vaccination with one dose of PR1 and WT1 peptides induces transient anti-leukemia immunity. We hypothesized that maintenance of a sustained anti-leukemia response may require frequent boost injections.

Design and Methods

Eight patients with myeloid malignancies were enrolled in this phase II study, and 6 completed 6 injections of PR1 and WT1 peptides in Montanide-adjuvant with GM-CSF, every two weeks.

Results

Both high- and low-avidity PR1 or WT1-specific CD8⁺ T cells were detected in all evaluable patients after the first vaccine dose. Repeated vaccination led to selective deletion of high avidity PR1- and WT1-specific CD8⁺ T cells and was not associated with significant reduction in WT1-expression. Additional boosting failed to increase vaccine-induced CD8⁺ T-cell frequencies further and in all patients the response was lost before the 6^th dose. PR1- or WT1-specific CD8⁺ T cells were not detected in bone marrow samples, excluding their preferential localization to this site. Following a booster injection three months after the 6^th vaccine dose, no high-avidity PR1 or WT1-specific CD8⁺ T cells could be detected, whereas low-avidity T cells were readily expanded.

Conclusions

These data support the immunogenicity of PR1 and WT1 peptide vaccines. However, repeated delivery of peptides with Montanide-adjuvant and GM-CSF leads to rapid loss of high-avidity peptide-specific CD8⁺ T cells. These results may offer an explanation for the lack of correlation between immune and clinical responses observed in a number of clinical trials of peptide vaccination. New approaches are needed to induce long-term high-avidity memory responses against leukemia antigens. (ClinicalTrials.gov Identifier: NCT00499772)     

Expression of TIM-3, Human β-defensin-2, and FOXP3 and Correlation with Disease Activity in Pediatric Crohn’s Disease with Infliximab Therapy

Background/Aims

This study investigated the expression of T cell immunoglobulin- and mucin-domain-containing molecule 3 (TIM-3), human β-defensin (HBD)-2, forkhead box protein 3 (FOXP3), and the frequency of CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells (Tregs) in children with Crohn’s disease (CD) during infliximab therapy.

Methods

We enrolled 20 CD patients who received infliximab treatment for 1 year. Peripheral blood and colonic mucosal specimens were collected from all CD patients and from healthy control individuals.

Results

A significant difference in TIM-3 mRNA expression was evident in peripheral blood mononuclear cells and colonic mucosa between CD patients before infliximab therapy and the healthy controls (p<0.001 and p=0.005, respectively). A significant difference in HBD-2 mRNA expression was found in colonic mucosa between CD patients before infliximab therapy and the healthy controls (p=0.013). In the active phase of CD, at baseline, the median percentage of T cells that were CD25⁺ FOXP3⁺ was 1.5% (range, 0.32% to 3.49%), which increased after inflixmab treatment for 1 year to 2.2% (range, 0.54% to 5.02%) (p=0.008).

Conclusions

Our study suggests that both the adaptive and innate immune systems are closely linked to each other in CD pathogenesis. And the results of our study indicate that it could be a useful therapeutic tool, where restoration of TIM-3, HBD-2 and the function of Tregs may repair the dysfunctional immunoregulation in CD.     

��-tocotrienol protects mouse and human hematopoietic progenitors from ��-irradiation through extracellular signal-regulated kinase/mammalian target of rapamycin signaling

Background

Exposure to γ-radiation causes rapid hematopoietic cell apoptosis and bone marrow suppression. However, there are no approved radiation countermeasures for the acute radiation syndrome. In this study, we demonstrated that natural δ-tocotrienol, one of the isomers of vitamin E, significantly enhanced survival in total body lethally irradiated mice. We explored the effects and mechanisms of δ-tocotrienol on hematopoietic progenitor cell survival after γ-irradiation in both in vivo and in vitro experiments.

Design and Methods

CD2F1 mice and human hematopoietic progenitor CD34⁺ cells were treated with δ-tocotrienol or vehicle control 24 h before or 6 h after γ-irradiation. Effects of δ-tocotrienol on hematopoietic progenitor cell survival and regeneration were evaluated by clonogenicity studies, flow cytometry, and bone marrow histochemical staining. δ-tocotrienol and γ-irradiation-induced signal regulatory activities were assessed by immunofluorescence staining, immunoblotting and short-interfering RNA assay.

Results

δ-tocotrienol displayed significant radioprotective effects. A single injection of δ-tocotrienol protected 100% of CD2F1 mice from total body irradiation-induced death as measured by 30-day post-irradiation survival. δ-tocotrienol increased cell survival, and regeneration of hematopoietic microfoci and lineage⁻/Sca-1⁺/ckit⁺ stem and progenitor cells in irradiated mouse bone marrow, and protected human CD34⁺ cells from radiation-induced damage. δ-tocotrienol activated extracellular signal-related kinase 1/2 phosphorylation and significantly inhibited formation of DNA-damage marker γ-H2AX foci. In addition, δ-tocotrienol up-regulated mammalian target of rapamycin and phosphorylation of its downstream effector 4EBP-1. These alterations were associated with activation of mRNA translation regulator eIF4E and ribosomal protein S6, which is responsible for cell survival and growth. Inhibition of extracellular signal-related kinase 1/2 expression by short interfering RNA abrogated δ-tocotrienol-induced mammalian target of rapamycin phosphorylation and clonogenicity, and increased γ-H2AX foci formation in irradiated CD34⁺ cells.

Conclusions

Our data indicate that δ-tocotrienol protects mouse bone marrow and human CD34⁺ cells from radiation-induced damage through extracellular signal-related kinase activation-associated mammalian target of rapamycin survival pathways.     

Differential effects of rapamycin and retinoic acid on expansion,stability and suppressive qualities of human CD4+CD25+FOXP3+ T regulatory cell subpopulations

Adoptive transfer of ex vivo expanded CD4⁺CD25⁺FOXP3⁺ regulatory T cells is a successful therapy for autoimmune diseases and transplant rejection in experimental models. In man, equivalent manipulations in bone marrow transplant recipients appear safe, but questions regarding the stability of the transferred regulatory T cells during inflammation remain unresolved. In this study, protocols for the expansion of clinically useful numbers of functionally suppressive and stable human regulatory T cells were investigated. Regulatory T cells were expanded in vitro with rapamycin and/or all-trans retinoic acid and then characterized under inflammatory conditions in vitro and in vivo in a humanized mouse model of graft-versus-host disease. Addition of rapamycin to regulatory T-cell cultures confirms the generation of high numbers of suppressive regulatory T cells. Their stability was demonstrated in vitro and substantiated in vivo. In contrast, all-trans retinoic acid treatment generates regulatory T cells that retain the capacity to secrete IL-17. However, combined use of rapamycin and all-trans retinoic acid abolishes IL-17 production and confers a specific chemokine receptor homing profile upon regulatory T cells. The use of purified regulatory T-cell subpopulations provided direct evidence that rapamycin can confer an early selective advantage to CD45RA⁺ regulatory T cells, while all-trans retinoic acid favors CD45RA⁻ regulatory T-cell subset. Expansion of regulatory T cells using rapamycin and all-trans retinoic acid drug combinations provides a new and refined approach for large-scale generation of functionally potent and phenotypically stable human regulatory T cells, rendering them safe for clinical use in settings associated with inflammation.     

Regulatory Role of CD4+CD25+Foxp3+ Regulatory T Cells on IL-17-Secreting T Cells in Chronic Hepatitis B Patients

Background

Both interleukin (IL)-17-secreting CD4⁺ T (Th17) and CD4⁺CD25⁺Foxp3⁺ T regulatory (Treg) cells have been shown to be associated with disease progression or liver damage in chronic hepatitis B (CHB) patients. However, the relationship between Treg cells and IL-17-secreting T cells in hepatitis B virus (HBV) infections is unclear.

Methods

The frequencies of Treg cells and IL-17-secreting T cells in hepatitis B e antigen (HBeAg)-positive CHB patients and healthy subjects were measured by flow cytometric analysis. The role of Treg cells on the differentiation of Ag-specific IL-17-secreting T cells was determined by removing the Treg cells from peripheral blood mononuclear cells (PBMCs) in HBeAg-positive CHB patients.

Results

The frequencies of both Th17 (1.71 ± 0.58 vs. 1.08 ± 0.36 %; P < 0.01) and Treg cells (8.92 ± 4.11 vs. 6.45 ± 1.56 %; P < 0.01) were increased in the peripheral blood of HBeAg-positive CHB patients compared with healthy controls, but in not the IL-17-secreting CD8⁺ T (Tc17) cells. The frequency of Treg cells was significantly associated with that of Th17 cells (r = 0.625, P = 0.001) in CHB patients. Spearman analysis showed a positive correlation between the frequency of Treg cells and HBV DNA load (r = 0.508, P = 0.008), as well as between the frequency of Th17 cells and serum alanine aminotransferase level (r = 0.516, P = 0.007). The deletion of Treg cells significantly enhanced both Th17 and Tc17 cell development in PBMCs following recombinant HBV core antigen stimulation.

Conclusions

Our data indicate a clear inverse relationship between Th17 cells and Treg cells and that Treg cells can inhibit Th17 cell development in CHB patients.     

Impact of constitutional polymorphisms in VCAM1 and CD44 on CD34+ cell collection yield after administration of granulocyte colony-stimulating factor to healthy donors

Background

The number of CD34⁺ cells mobilized from bone marrow to peripheral blood after administration of granulocyte colony-stimulating factor varies greatly among healthy donors. This fact might be explained, at least in part, by constitutional differences in genes involved in the interactions tethering CD34⁺ cells to the bone marrow.

Design and Methods

We analyzed genetic characteristics associated with CD34⁺ cell mobilization in 112 healthy individuals receiving granulocyte colony-stimulating factor (filgrastim; 10 μg/kg; 5 days).

Results

Genetic variants in VCAM1 and in CD44 were associated with the number of CD34⁺ cells in peripheral blood after granulocyte colony-stimulating factor administration (P=0.02 and P=0.04, respectively), with the quantity of CD34⁺ cells ×10⁶/kg of donor (4.6 versus 6.3; P<0.001 and 7 versus 5.6; P=0.025, respectively), and with total CD34⁺ cells ×10⁶ (355 versus 495; P=0.002 and 522 versus 422; P=0.012, respectively) in the first apheresis. Of note, granulocyte colony-stimulating factor administration was associated with complete disappearance of VCAM1 mRNA expression in peripheral blood. Moreover, genetic variants in granulocyte colony-stimulating factor receptor (CSF3R) and in CXCL12 were associated with a lower and higher number of granulocyte colony-stimulating factor-mobilized CD34⁺ cells/μL in peripheral blood (81 versus 106; P=0.002 and 165 versus 98; P=0.02, respectively) and a genetic variant in CXCR4 was associated with a lower quantity of CD34⁺ cells ×10⁶/kg of donor and total CD34⁺ cells ×10⁶ (5.3 versus 6.7; P=0.02 and 399 versus 533; P=0.01, respectively).

Conclusions

In conclusion, genetic variability in molecules involved in migration and homing of CD34⁺ cells influences the degree of mobilization of these cells.